Mechanisms of the Ershiwuwei Guijiu Pill in Treating Postmenopausal Osteoporosis Based on Network Analysis and Experimental Validation

BackgroundThe Tibetan medicine Ershiwuwei Guijiu Pill (EWGP), a classic Tibetan medicine prescription for the treatment of postmenopausal osteoporosis (PMOP) in the Qinghai-Tibet region, has attracted extensive attention due to its curative effects on gynecological diseases. However, its chemical ingredients and molecular mechanism are still unclear.Aim of the studyTo analyze the chemical constituents and effective serum chemical metabolites of EWGP and to explore the molecular mechanism of EWGP in treating PMOP through network analysis and experimental validation.MethodsThe ethanol extract of EWGP and its drug-containing serum were detected by liquid chromatography-mass spectrometry (LC-MS), and the chemical constituents were analyzed and identified. SwissTarget prediction was used to predict the corresponding potential target genes of the identified chemical components. Thereafter, a visualization network of the components and corresponding targets was constructed with Cytoscape software. Moreover, a specific disease database for animals was used to search and filter for osteoporosis (OP) targets, and a drug-disease target protein-protein interaction (PPI) network was constructed. Cytoscape 3.7.0 was used for visualization and cluster analysis, and R Studio was used for GO and KEGG enrichment analysis. AutoDock Tools were applied for molecular docking of the serum metabolites and specific target proteins. The potential mechanism of EWGP in preventing and treating PMOP was predicted by network pharmacology analysis and was experimentally studied and verified <i>in vivo</i> and <i>in vitro</i>.ResultsA total of 199 chemical substances were identified in the ethanol extract, and 11 were found in the serum. A total of 419 predicted targets and 128 target genes related to osteoporosis were screened. There were 16 common targets identified between the predicted targets and OP genes. Following the enrichment analysis, 16 KEGG signaling pathways and 63 GO biological process items were identified. The results of molecular docking showed that the main active compounds may be Protopine, Hetisine, Piperine, Visaminol, Boldine, and Trigonelline, and the specific targets may be CYP17A1, ESR2, MAPK14, and the vitamin D receptor (VDR). The results of cell and animal experiments showed that EWGP may improve bone metabolism via estrogen and calcium signaling pathways regulated by estrogens and calcium ions.ConclusionsEWGP contains multiple herbal drugs and treats PMOP through multiple targets and signaling pathways. We preliminarily tested the chemical compounds of EWGP, especially in the serum, to determine the chemical metabolites of EWGP and revealed the molecular mechanism of EWGP in preventing and treating PMOP; moreover, we used computer-virtual molecular docking and experiments for preliminary verification of the core targets of network pharmacology analysis.